Synthetic Study toward Total Synthesis of (±)-Germine: Synthesis of (±)-4-Methylenegermine.

The total synthesis of 4-methylenegermine is described.

Radiochemical synthesis and in vivo evaluation of [18F]AZ11637326: an agonist probe for the α7 nicotinic acetylcholine receptor.

INTRODUCTION: The alpha-7 nicotinic acetylcholine receptor (α7 nAChR) is key in brain communication and has been implicated in the pathophysiology of diseases of the central nervous system. A positron-emitting radioligand targeting the α7 nAChR would enable better understanding of a variety of neuropsychiatric illnesses, including schizophrenia and Alzheimer's disease, and could enhance the development of new drugs for these and other conditions. We describe our attempt to synthesize an α7 nAChR-selective radiotracer for positron emission tomography (PET). METHODS: We prepared the high-affinity (K(d) = 0.2 nM) α7 nAChR agonist, 5'-(2-[(18)F]fluorophenyl)spiro[1-azabicyclo-[2.2.2]octane]-3,2'-(3'H)furo[2,3-b]pyridine, [(18)F]AZ11637326, in two steps, a nucleophilic fluorination followed by decarbonylation. We studied [(18)F]AZ11637326 in rodents, including mice lacking α7 nAChR, and in non-human primates. RESULTS: [(18)F]AZ11637326 was synthesized in a non-decay-corrected radiochemical yield of 3% from the end of synthesis (90 min) with a radiochemical purity >90% and average specific radioactivity of 140GBq/µmol (3,781 mCi/µmol). Modest rodent brain uptake was observed (2-5% injected dose per gram of tissue, depending on specific activity), with studies comparing CD-1 and α7 nAChR null mice indicating an element of target-specific binding. Blocking studies in non-human primates did not reveal specific binding within the brain. CONCLUSION: Despite the high affinity and target selectivity of AZ11637326 for α7 nAChR in vitro and encouraging rodent studies, receptor-mediated binding could not be demonstrated in non-human primates. Further structural optimization of compounds of this class will be required for them to serve as suitable radiotracers for PET.

In vitro binding characteristics of [3H]AZ11637326, a novel alpha7-selective neuronal nicotinic receptor agonist radioligand.

AZ11637326 (5'-(2-fluoro[3,4,5(-3)H3]phenyl)-spiro[1-azabicyclo [2.2.2]octane-3,2'(3'H)-furo[2,3-b]pyridine) is a potent partial agonist at the human alpha7 neuronal nicotinic receptor with sub-nanomolar affinity for the human and rat alpha7 [(125)I]alpha-bungarotoxin binding sites. In a search for novel agonist radioligands and imaging ligands for the alpha7 nicotinic receptor, [(3)H]AZ11637326 was synthesized and its in vitro membrane binding properties were characterized. [(3)H]AZ11637326 bound to halpha7-HEK membranes with high specificity (>95%), high affinity (230 pM) and a B(max) of 460 fmol/mg. The rank order of affinity of nicotinic standards determined with [(3)H]AZ11637326 strongly correlated with those determined using the classical alpha7 antagonist [(125)I]alpha-bungarotoxin, indicating that [(3)H]AZ11637326 bound to halpha7-HEK membranes with an alpha7 nicotinic-like pharmacology. The K(i) values for the standards were on average 2.3-fold lower affinity than determined using the prototypical alpha7 nicotinic antagonist [(125)I]alpha-bungarotoxin. Because [(3)H]AZ11637326 specific binding is rapid and reversible, the K(i) values determined using this ligand are more accurate estimates of affinity than those determined using the kinetically sluggish [(125)I]alpha-bungarotoxin. [(3)H]AZ11637326 also bound to a high affinity (510 pM), nicotine-sensitive site on rat hippocampal membranes with an average B(max) of 55 fmol/mg. With rat hippocampal membranes, the nicotine-sensitive fraction of total binding was sub-optimal for a radioligand (~50%), yet the potencies and rank order of the K(i) values for standards were consistent with an alpha7 nicotinic pharmacology. Overall, these studies indicate that [(3)H]AZ11637326 is a useful new in vitro probe of the alpha7 nicotinic receptor agonist site and support its potential utility for in vivo receptor occupancy studies.

Synthesis and biodistribution of radiolabeled alpha 7 nicotinic acetylcholine receptor ligands.

UNLABELLED: Our objective was to develop an array of alpha(7)-selective nicotinic cholinergic receptor (nAChR)-based imaging agents for PET and SPECT. METHODS: (2'R)-N-(11)C-Methyl-N-(phenylmethyl)-spiro[1-azabicyclo[2.2.2]octane-3,2'(3'H)-furo[2,3-b]pyridin]-5'-amine 1 was synthesized by reaction of the corresponding desmethyl precursor with (11)C-CO(2) and reduction. N-(R)-1-Aza-bicyclo[2.2.2]oct-3-yl-4-(11)C-methylsulfanyl-benzamide 2 was synthesized by reduction of the corresponding disulfide precursor and reaction with (11)C-iodomethane. N-(R)-1-Aza-bicyclo[2.2.2]oct-3-yl-4-(125)I-iodo-benzamide 3 was synthesized by halogen exchange of the corresponding bromide. (2'R)-5'-(2-(125)I-iodo-3-furanyl)spiro[1-azabicyclo[2.2.2]octane]-3,2'(3'H)-furo[2,3-b]pyridine 4 was synthesized by the chloramine-T method. Kinetic biodistribution studies were done in male CD-1 mice by tail vein injection of 3.7 MBq (100 microCi) of the (11)C-labeled radiotracer or 0.67 MBq (2 microCi) of the (125)I-labeled radiotracer followed by brain dissection and tissue counting. Receptor blockade was determined by pretreatment of the mice with an excess of either unlabeled precursor or nicotine. RESULTS: We synthesized 4 radiolabeled, moderate- to high-affinity, alpha(7)-nAChR-based ligands. The compounds were a series of quinuclidine derivatives with an inhibition constant (K(i)) < 6 nmol/L (33 pmol/L for 4) for alpha(7)-nAChR and selectivities of alpha(7)/alpha(4)beta(2) subtypes of > or =14,000. All of the compounds were produced in adequate radiochemical yield and specific radioactivity (>74 GBq/micromol [2,000 Ci/mmol]). No site selectivity or receptor blockade was shown for 1 and 2 (0.91 +/- 0.05 and 0.14 +/- 0.03 %ID/g [percentage injected dose per gram] in the hippocampus [target tissue], respectively). Compound 3 showed low hippocampal uptake (0.25 +/- 0.05 %ID/g) but prolonged retention within that structure. Pretreatment with nicotine decreased its uptake by up to 50% in the hippocampus. Similar reductions were also observed within the cerebellum (nontarget tissue). Compound 4 showed hippocampal uptake of 2.41 +/- 0.03 %ID/g and target-to-nontarget uptake ratios of up to 2. Pretreatment of animals with unlabeled 4 resulted in a decrease of hippocampal uptake to 60% of its preblockade value without a corresponding decrease in cerebellar uptake. CONCLUSION: With further structural optimization, selective imaging of alpha(7)-nAChR may be possible.

[3 + 2] cycloaddition reactions of 4-alkyl-3-hydroxy-2H-pyrazolo[4,3-c]isoquinolinium inner salts.

Heating dipolarophiles with 4-alkyl-3-hydroxy-2H-pyrazolo[4,3-c]isoquinolinium hydroxide inner salts results in [3 + 2] cycloaddition across positions 3a and 5 of the aromatic system to give the [3 + 2] cycloadducts in good yield. When the 4-alkyl substituent is a 2-acetate ester and the methylene group can be deprotonated, a second mode of [3 + 2] cycloaddition becomes available for the resulting anion (across the side chain methine group and position 5 of the aromatic system) and occurs under basic conditions, allowing either of two modes of [3 + 2] cycloaddition to be selected by appropriate choice of reaction conditions.

Synthesis of methyl (5S,6R,7E,9E,11Z,13E,15S)-16-(4-fluorophenoxy)-5,6,15-trihydroxy-7,9,11,13-hexadecatetraenoate, an analogue of 15R-lipoxin A4.

We describe a method for the synthesis of methyl (5S,6R,7E,9E,11Z,13E,15S)-16-(4-fluorophenoxy)-5,6,15-trihydroxy-7,9,11,13-hexadecatetraenoate, a compound that has been described as a metabolically stable analogue of 15R-lipoxin A(4).